Suprabasal breast cell with stem cell properties

ABSTRACT

A method for isolating cells which have a suprabasal position and expresses epithelial specific antigen but no sialomucin is provided. The isolated cells are shown to share many of the properties expected of a mammary gland stem cell. Three permanent cell lines that are capable of proliferating and capable of differentiating into cells of mammary gland luminal epithelial and myoepithelial cell lineages were established. Such cells form elaborate branching structures resembling uncultured terminal duct lobular units both by morphology and marker expression both in vitro and in vivo. Evidence is provided that these keratin K19 expressing cells most probably is the cells in which breast cancer arises. Thus they constitute a model not only for the developing breast, but also for the development of breast cancer. Also disclosed are the uses of such isolated cells or the cell lines as a model system of the mammary gland for pharmacological studies and their uses in tissue repair or transplantation.

FIELD OF INVENTION

The present invention relates to the isolation of a new at leastbi-potent cell type from luminal epithelial cells of a mammary gland andits establishment as an immortalised cell line which is capable ofproliferating and capable of differentiating into cells of mammary glandluminal epithelial and myoepithelial cell lineages. The inventionfurthermore relates to the uses of the isolated cells or the cell linesas a model system of the mammary gland and to the uses in tissue repairor transplantation.

GENERAL BACKGROUND

Understanding how the normal human breast develops and which cellcompartment becomes neoplastic by necessity is dependent on theisolation of relevant cells as the true targets of human breastcarcinogenesis and progression. More than two decades ago it wasproposed that human breast cancer originates from the luminal epitheliallineage within the terminal duct lobular units (TDLU), a basic mammarystructure consisting of a branching ductal-alveolar system lined by aninner layer of luminal epithelial cells and an outer layer ofmyoepithelial cells.

Recently, others and the present inventors have provided some evidencethat the stem cells of the human and mouse breast gland may be containedwithin the luminal epithelial lineage (Smith 1996; Stingl et al. 1998;Péchoux et al. 1999; Smalley et al. 1999; Stingl et al. 2001). However,detailed ultrastructural characterisations of the rodent and humanbreast gland in situ have led to the widely discussed hypothesis thatthe stem cell is a “basal cell” with clear cytoplasm (for review see(Smith and Chepko 2001). In particular, the important work of Smith andcolleagues in the mouse mammary gland has outlined an elaboratemorphological tree identifying the actual stem cell (small light cells;SLC), a first degree progenitor cell (not distinct from SLC), a seconddegree progenitor which is still multipotent (undifferentiated largelight cell, ULLC), and then two compartments of non-dividing,pre-luminal- and pre-myoepithelial cells which gradually mature into thefully differentiated lineages. At the ultrastructural level, the SLCnever reaches the acinus lumen and only a fraction of the ULLC does so(Smith and Chepko 2001). Since sialomucin is the most prominent markerof luminal epithelial cells and is exclusively expressed on the apicalsurface of luminal epithelial cells, this would imply that someepithelial cells (not facing the lumen) are sialomucin negative.Nevertheless, there is reason to believe that these cells are indeedfull members of the luminal epithelial lineage. The cells on the basalside of multilayered breast ducts express several luminal epithelialmarkers including simple epithelial keratins and epithelial specificantigen (ESA), but no markers of the myoepithelial lineage such asα-smooth muscle actin or Common Acute Lymphoblastic Leukaemia Antigen(CALLA). Cells with this phenotypic profile have in fact been observedin cultures of human reduction mammoplasties and shown to bebi-potent—suggesting a stem cell potential (Stingl et al. 1998).However, further characterisation of the putative stem cells to show thefull potential of generating TDLU have not been pursued due to a limitedgrowth potential in primary culture.

SUMMARY OF THE INVENTION

The problem to be solved by the present invention was to isolate thecells of the mammalian breast gland which are able to form terminal ductlobular units (TDLUs) in order to provide a cell culture which may serveas a model for breast gland development. Since breast cancer issuspected to originate in the cells forming the TDLUs such cells could,if isolated, also serve as a model for breast cancer development.

The solution was to define and then to isolate such a cell type. Basedon the literature and our own extensive observations the presentinventors reasoned that a breast epithelial cell type which expressesepithelial specific antigen (ESA) but little or no sialomucin seemed agood candidate to the precursor cell forming TDLU.

The present inventors show that a minor population ofsuprabasal-positioned, subluminal, ESA-positive andsialomucin-negative/weakly positive cells (ESA⁺/MUC⁻ cells) indeedexists in vivo.

To accomplish the isolation of such a cell, the present inventorsisolated distinct cell populations using immunomagnetic sorting by firstremoving the sialomucin expressing cells and then isolatingsialomucin-negative cells that expresses ESA. The present Inventors showthat the disclosed method of isolation consistently results in cellsthat possess properties expected of TDLU precursor cells. Accordingly,the invention provides in a first aspect a method for isolation of an atleast bi-potent mammary gland tissue cell, comprising the steps of:

(i) separating said tissue into two or more different cell types

(ii) culturing each of said different cell types under celldifferentiation conditions and

(iii) selecting the cell type(s) that is/are capable of differentiatinginto at least two morphologically and/or phenotypically different celltypes.

The isolated suprabasal-derived ESA⁺/MUC⁻ cells were of luminalepithelial lineage because they expressed tight junction proteins andexhibited a high transepithelial electrical resistance on transwellfilters. However, in contrast to luminal epithelial cells with strongsialomucin expression they had a striking ability to form the entireTDLUs inside a three-dimensional reconstituted basement membrane and innude mice and could generate myoepithelial cells, as well as luminalepithelial cells. Thus these cells share many of the properties expectedof a mammary stem cell. Accordingly, an important aspect of theinvention is to provide an isolated cell, derived from luminalepithelial cells of a mammary gland, which is capable of proliferatingand capable of differentiating into cells of mammary gland luminalepithelial and myoepithelial cell lineages. In a further aspect, theinvention pertains to a cell population composed of such cells.

In further aspects of the invention, immortalised cell lines derivedfrom said isolated cells that are capable of differentiating into cellsof mammary gland luminal epithelial and myoepithelial cell lineages areprovided.

To obtain such permanent cell lines with stem cell properties the cellswere immortalised with HPV (human papilloma virus)-16 E6/E7. Three celllines that are capable of proliferating and capable of differentiatinginto cells of mammary gland luminal epithelial and myoepithelial celllineages were established (D492, D490 and TH69). One of these (D492) wasdeposited in accordance with the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Micro-organisms for thePurposes of Patent Procedure at Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH (DSMZ) and has obtained the accession number DSMACC 2529. Also non-immortalised cell suprabasal-derived epithelial cellcultures obtained by the disclosed method of isolation (TH82 and TH95)possess stem cell properties.

Importantly, suprabasal-derived ESA⁺/MUC⁻ cells differ fromluminal-derived ESA⁺/MUC⁺ cells by the expression of keratin K19. Thepresent inventors have localised a subpopulation of luminal epithelialcells in the normal breast in situ by the restricted expression ofkeratin K19 similarly to the cells of the invention. The presentinventors propose that these cells are indeed candidate stem cells ormultipotent progenitor cells of the mammalian breast gland. Thus animportant embodiment of the invention is an immortalised cell line thatis derived from said isolated cells that are capable of differentiatinginto cells of mammary gland luminal epithelial and myoepithelial celllineages and which comprises cells that are positive staining forkeratin K19.

To the best of our knowledge the invention is the first in vitro modelof the developing human breast gland model.

The present inventors propose that, since more than 90% of human breastcarcinomas are keratin K19 positive and originate from TDLU, the presentinventors have identified the cellular origin of most human breastcancers. Thus a further aspect of the invention is to use the at leastbi-potent breast cells of the invention, and which shares many of thecharacteristics of the putative cellular origin of most human breastcancers, as an in vitro model for the study of breast cancer developmentand in particular to provide a method for testing the carcinogeniceffect, if any, of a substance on mammary gland epithelial cells, themethod comprising:

(i) culturing said cells in a growth medium which maintains the cells asnon-transformed cells;

(ii) adding the agent, compound or factor under test to the cellculture; and

(iii) determining the neoplastic response, if any, of the so contactedcells by changes in morphology, tumorigenicity in animals, mRNAexpression and/or antigen expression as well as other changes which isassociated with carcinogenicy.

In further aspects of the invention the isolated suprabasal-derivedESA⁺/MUC⁻ cells are used as an in vitro model of the developing humanbreast gland to screen for pharmaceutical interesting or toxicsubstances. Thus the invention provide a method for testing the toxiceffect, if any, of a substance on mammary gland epithelial cells,comprises:

(i) culturing or maintaining said cells in a non-toxic medium;

(ii) adding the substance to be tested to the medium; and

(iii) determining the response, if any, of the cells, including changesin cell growth rate, cell death rate, apoptosis, cell metabolism, inter-as well as intra-cellular communication, morphology, mRNA or proteinexpression and antigen expression.

In further aspects the cells of the invention are used to providemutatis mutandis a similar three step method for testing the ability, ifany, of a substance to modulate the differentiation of non-terminaldifferentiated mammary gland epithelial cells and a three step methodfor screening a substance for its ability, if any, to interact with acellular protein.

In a further aspect of the invention the cells isolated by the methoddisclosed in the present application are expanded and provides a methodof transplanting a vertebrate host with said cells, comprising the stepof introducing the cell into the vertebrate host. In yet a furtherdevelopment of this aspect said cells provide a method of in vivoadministration of a protein or gene of interest to an individual in needthereof, comprising the step of transfecting the cell-population with avector comprising DNA or RNA which expresses the protein or gene ofinterest and introducing the transfected cell into said individual. Inyet a further development of the invention said cells provide a methodof tissue repair or transplantation in mammals, comprising administeringto a mammal a therapeutically effective amount of cells or tissuesderived therefrom.

DETAILED DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a cell culture, whichis able to form a structure similar to the terminal duct lobular units(TDLUs) of the mammary gland in order to provide a cell culture, whichserves as a model for the normal breast gland development. In thepresent context the “terminal duct lobular units (TDLUs)” of the mammarygland is defined as the basic mammary structure consisting of abranching ductal-alveolar system lined by an inner layer of luminalepithelial cells and an outer layer of myoepithelial cells.

Based on experiments with cells in culture it has been suggested that aputative “stem” cell of the human TDLUs may be contained within theluminal epithelial lineage of cells and characterised by a positivestaining for the luminal epithelial marker (ESA⁺) and a negative orweakly positive staining for sialomucin (MUC⁻). In Example 1 data ispresented showing that such an MUC⁻/ESA⁺ luminal epithelial cell typeexists in the human breast in vivo and that they are suprabasallypositioned.

Example 2 describes the isolation, immortalisation and characterisationof suprabasal-derived epithelial “stem” or “progenitor” cells as well asthe isolation of luminal cells without “stem-cell” properties. In thepresent context “stem cell” or “progenitor cell” is defined as an atleast bi-potent mammary gland tissue cell that is able to differentiateinto cells of mammary gland luminal epithelial and myoepithelial celllineages. The progenitor cells were isolated the fromsuprabasal-positioned luminal epithelial cells of the mammary gland byimmunomagnetic cell sorting exploiting the assumed MUC⁻/ESA⁺ phenotypeof the wanted cell type.

In the present context the term “suprabasal” is defined as abluminal andseparated from the basement membrane by a layer of myoepithelial cells.The term “suprabasal-positioned” is used to describe cells that arepositioned between the luminal and the myoepithelial cell layer, and theterm “suprabasal-derived” is used to describe cells that are isolatedfrom the suprabasal layer of luminal cells of the TDLUs.

In a particular useful embodiment of the invention an isolated cell,derived from luminal epithelial cells of a mammary gland, which iscapable of proliferating and capable of differentiating into cells ofmammary gland luminal epithelial and myoepithelial cell lineages andwhich is isolated from suprabasal luminal epithelial cells of themammary gland is provided.

In one embodiment of the invention, which is illustrated in example 2,the isolated cell is a human cell. It is however contemplated that asimilar stem or progenitor cell also can be isolated by the proceduredescribed in example 2 from mamma of other species resulting in a cellselected from the group consisting of a rodent cell, a porcine cell, aruminant cell, a bovine cell, a caprine cell, a equine cell, a caninecell, a ovine cell, a feline cell and a primate cell.

As illustrated in example 2 such an isolated cell is capable of forminga cell culture comprising cells which are positive staining for theluminal epithelial marker ESA (ESA+) and negative or weakly positivestaining for sialomucin (MUC−), so-called ESA⁺/MUC− cells. By “positivestaining” the present inventors refer to clearly visible cells afterstaining with the primary antibody in a 1:200 dilution forimmunoperoxidase or a 1:10 dilution for immunofluorescence.

In a preferred embodiment of the invention said isolated cell isimmortalised. An immortalised cell is a prerequisite for establishing apermanent cell line. Whereas rodent cells are relative prone to undergoeven apparently spontaneous immortalisation human cells are remarkablyresistant to immortalisation. Thus a particular useful embodiment of thepresent invention is an immortalised ESA+/MUC− cell. However cells,including human cells, may be immortalised by a number of procedures.The literature provides examples of cells being immortalised as a resultof exposure to various chemicals including carcinogens and tumourpromoters (Balmain and Harris, 2000) and as a result of the introductionof a nucleic acid molecule encoding an immortalising polypeptide(Katakura et al., 1998). By “a nucleic acid molecule encoding animmortalising polypeptide” the present inventors refer to a nucleic acidmolecule that codes for a polypeptide the expression of which eitheralone or in combination with other polypeptides result in theimmortalisation of the respective cell. In the present context animmortalised cell is defined as a cell capable of in vitro growth forpreferably at least 50 doublings, more preferably at least 75 doublings,and most preferably at least 100 doublings. This is to be compared withthe normal situation where senescence occurs after 30 doublings.Furthermore a distinct telomerase activity which is absent from finitelife span breast epithelial cells can be used to define immortalisedcells (Stampfer et al, 2001). Thus one specific embodiment of theinvention is an immortalised cell line, wherein the immortalising stepcomprises transfecting the cells with a nucleic acid molecule encodingan immortalising polypeptide.

According to presently preferred embodiment, the immortalisation isperformed by introducing into the cells a nucleic acid vector comprisingat least one nucleic acid sequence encoding an oncogenic polypeptideselected from the group of transforming oncogenes which has been shownto be able to immortalise cells either alone or in combination withother genes. Examples of such genes are c-myc, N-myc, L-myc, SV40 largeT antigen, adenovirus E1A, papillomaviruses E6 and E7, polyoma Large Tgene, erbA, myb, fos, jun, p53 or an oncogenic part of any one thereof.However, also nucleotide sequences from Epstein-Barr virus, Herpes virusand certain other virus has been implicated in the immortalisation ofcells.

Recently, a number of reports have demonstrated the immortalisationpotential of human papillomavirus-16 E6 and E7 genes (HPV16-E6/E7). Thusaccording to an embodiment of the invention, immortalised cell line ischaracterised by an immortalising step that comprises transfecting thecells with a nucleic acid molecule encoding a papillomavirus polypeptideselected from the group consisting of E6, E7 and a nucleic acid moleculecomprising E6 and E7.

The major concern with this technique is that while it has been shownthat immortality is achieved by the inactivation of p53 andretinoblastoma protein (RB), these may not be the only affectedmolecules and other cellular functions may also be affected (for reviewsee (Zwerschke and Jansen-Durr 2000). Evidence suggests, however, thathuman cells derived from E6/E7 immortalisation retain much of theiroriginal phenotype. In organotypic cultures of endocervical cells, whichis a target organ in vivo for HPV-16 infection, the cells appearednormal with ordinary stratification and production of a cornified layer.Also, normal adult human pancreatic epithelial cells transfected withE6/E7 remained polarized on collagen gels did not grow in soft agar andexpressed typical simple keratins. Thus, whereas E6 and E7 readilyinduce a neoplastic transformation of rodent cells when transferred intothe cells, human cells appear to be significantly more robust. Humancells transfected with E6 and E7 do not form tumours in nude mice evenafter more than 100 passages in culture (Willey et al. 1991; Band1995;). Immortalisation of normal human breast cells with either E6 orE7 did not lead to aberrant functional behaviour in luminal ormyoepithelial cells tested (Wazer et al. 1995). It has been reportedthat breast cells lose keratin K19 expression as a consequence ofE7-immortalisation (Spancake et al. 1999), but this was not confirmed bythe present inventors (see below) or others (Wazer et al. 1995).Finally, human salivary gland cells transduced with E6/E7 remaineddiploid or near-diploid without a general destabilisation of thekaryotype. The present inventors have confirmed these studies, and havefound that the transduced cells are non-tumorigenic and have a diploidkaryotype (46, XX) even after more than one year in culture and 25passages. Thus it appears that the method used for immortalising theisolated MUC⁻/ESA⁺ progenitor cells and which comprises transforming thecells with a retroviral vector including an expression cassettecomprising a nucleic acid molecule encoding a papillomavirus polypeptideE6 and E7, and selecting the immortalised cells results in anon-tumorigenic cell line.

As used herein, the expressions “transforming” and “transducing” areused interchangeably and refer to the introduction of DNA into arecipient cell, irrespective of the method used for the introduction.Also the term “transfection” refers to the introduction of DNA into arecipient cell. But whereas the term “transduction” typically refers toa method of introduction which comprises virus particles, the term“transfection” may refer both to methods which involves virus as well asmethods which rely on virus-free compositions containing specificnucleic acids. In relation to eucaryotic cells the term “transfection”normally refers to the introduction of virus-free DNA compositions intoa recipient cell. In the present context the expressions “transforming”,“transduction”, and “transfection” are used interchangeably to describethe introduction of DNA into a recipient cell. Note however, that theterms “preneoplastic transformation” or “malignant transformation” ingeneral and also in the present context refer to the process wherein acell changes on or more phenotypically traits that characterise aneoplastic cell. One such trait is the ability to form tumours insyngeneic or immuno-incompetent animals.

A “vector” is a composition which can transduce, transform or infect acell, thereby causing the cell to express vector encoded nucleic acidsand, optionally, proteins other than those native to the cell, or in amanner not native to the cell. A vector includes a nucleic acid(ordinarily RNA or DNA) to be expressed by the cell. A vector optionallyincludes materials to aid in achieving entry of the nucleic acid intothe cell, such as a retroviral particle.

In a preferred embodiment of the invention the cells are immortalised bytransforming the cells with a retroviral vector. By the term “retroviralvector” are meant vectors that comprises retroviruses. Most retroviralvectors are based on murine retrovirus. They can carry 6 to 7 kb offoreign DNA (promoter+cDNA). Thus, according to the said preferredembodiment, the purified precursor cells were immortalised by aprocedure which comprises transforming the cells with at least oneretroviral vector including an expression cassette comprising a nucleicacid molecule encoding a papillomavirus polypeptide selected from thegroup consisting of E6, E7 and a nucleic acid molecule comprising E6 andE7, and selecting the immortalised cells.

The invention further relates to a method of immortalisation that isbased on other vectors for instance vectors based on adenovirus,adeno-associated virus, papilloma virus and plasmids.

In all cases the vector must comprise an expression cassette. By theterm “expression cassette” is meant a nucleic acid sequence thatcomprises the elements necessary to express an inserted cDNA in the hostof interest. For a mammalian cell host, such a vector typically containsa powerful promoter coupled to an enhancer, a cloning site, and apolyadenylation signal. In addition to the expression cassette, severalexpression vectors also contain a selectable marker gene such as DHFR orNeoR, which aids in the generation of stable cell lines. The expressioncassette may contain one or more unrelated DNA sequences encoding one ormore peptides of interest.

In a particular, the immortalising step can be performed by transformingthe cells with retrovirus-containing supernatant from the PA317 LXSNHPV16E6E7 packaging cell line (CRL-2203, ATCC, Rockville, Md.) cell lineand selecting the immortalised cells. Thus a preferred embodiment ofpresent invention is an immortalised cell line, wherein theimmortalising step is performed by transforming the cells withretrovirus-containing supernatant from the PA317 LXSN HPV16E6E7 cellline and selecting the immortalised cells.

As described in example 3 only the suprabasal-derived cell lines wereable to develop into mixed cultures of luminal epithelial andmyoepithelial cells.

The fact that the suprabasal-derived epithelial cell lines continued formore than 50 passages to generate subpopulations of ESA⁺/MUC⁺ andESA⁻/MUC⁻ cells as well as ESA⁺/MUC⁻ cells make them strong candidatesfor stem cells or multipotent progenitors of the breast, as theESA⁺/MUC⁺ cells represent differentiated luminal epithelial cells, whilethe ESA⁻/MUC⁻ cells represent myoepithelial cells, which are ESA⁻/MUC⁻in vivo.

In example 3 this hypothesis was further strenghtened by double-stainingclonal cultures for keratin K18 (luminal marker) and K14 (myoepithelialmarker). From these experiments it was clear that whereas theluminal-derived epithelial cell line did not generate any myoepithelialcells, the suprabasal-derived cell line readily formed mixed clones ofluminal epithelial and myoepithelial cells. This conclusion was furtheremphasised by the observation that the myoepithelial cells represented aprimitive level of myoepithelial differentiation because <1% of thecells expressed other myoepithelial markers such as Thy-1 andfurthermore that such Thy-1 expressing myoepithelial-like cells alsoexpressed α-smooth muscle actin which is restricted to postmitoticmyoepithelial cells in vivo (FIG. 3A, b, c) (Sapino et al. 1990).

In example 3 data are also presented showing that the K18⁺ cells werealso precursor cells of the lineage-restricted progeny within theluminal compartment. It is shown that these cells could further maturewithin this compartment to differentiated sialomucin-expressing cells.Taken together, these observations provide evidence for the existence ofa suprabasal, at least bi-potent epithelial cell belonging to theluminal epithelial lineage that can give rise to differentiatedmyoepithelial and luminal epithelial cells, and their precursors.

The perhaps most striking feature of the cells of the invention is thedemonstration in example 4 that an immortalised suprabasal-derivedepithelial cell line in culture is capable of forming branchingstructures resembling terminal duct lobular units of the mammary glandnot only by marker expression as demonstrated in example 3 but also inmorphology. In general, the criterion for stem cells of the breast istheir ability to regenerate the entire structure of the mammary gland.In mice and rats the ability to regenerate the entire structure of themammary gland upon reimplantation of cells in syngeneic gland-free fatpads has been adapted as the standard criteria for stem cells (Smith andMedina 1988). In example 4 a similar test in a laminin-rich gel wasperformed. When suprabasal-derived epithelial ESA+/MUC− cells wereembedded into the gel and cultured under these three-dimensional cultureconditions they gave rise to formation of larger, elaborate morphologiesresembling the entire functional unit of the breast gland, i.e. theterminal duct lobular unit (TDLU). In this respect thesuprabasal-derived epithelial cell-lines formed TDLU-like structures ata frequency (73%) similar to that recorded for suprabasal cells thatwere freshly prepared from primary cultures from two different biopsies.The TDLU-like structures were stained for differentiation markers ofnormal breast and revealed a remarkable similarity to TDLU's in vivo(FIG. 4D). Importantly, this was true for all the three cell lines(D492, D490 and TH69). The present inventors conclude that thesuprabasal-derived at least bi-potent cell lines posses many of thecharacteristics of a human progenitor cell of the breast gland, andaccordingly a preferred embodiment of the invention is an immortalisedcell line that in culture is capable of forming branching structuresresembling terminal duct lobular units of the mammary gland inmorphology and/or by marker expression.

As demonstrated in example 5 cells isolated, immortalised andestablished as a cell line according to the methods disclosed in thepresent invention comprises cells that are positively staining for thekeratin K19. It is considered a major novel and surprising conclusionthat the progenitor cells of the human breast reside in a keratin K19⁺compartment since it has been the technical prejudice within the fieldthat the potential stem cells of the breast were keratin K19⁻ cells.This was based on earlier observations that benign proliferative lesionsand highly proliferative breast cell lines were keratin K19⁻, and thatkeratin K19⁺ cells proliferate poorly in culture (Rønnov-Jessen et al.1996). However, more recent evidence supports our observation of akeratin K19⁺ precursor cell compartment. First, keratin K19 is one ofthe earliest keratins expressed in the embryo, and whereas the foetalbreast contains a homogeneously keratin K19⁺ luminal epithelialcompartment, keratin K19⁻ luminal cells arise only in adulthood(Anbazhagan et al. 1998). Second, more than 90% of breast carcinomas areK19⁺. While it could be argued that K19⁻ luminal epithelial cells couldturn on K19 along with malignant transformation, so far all malignant orpreneoplastic transformations of non-malignant K19⁻ breast cell lineshave resulted in K19⁻ tumour cells (Petersen et al. 1998; Santner et al.2001). Third, studies of other organs, including liver, pancreas, skin,testes, and prostate have revealed that the “stem cell compartment”express keratin K19 (Stosiek et al. 1990; Fridmacher et al. 1995; Michelet al. 1996; Bouwens 1998; Hudson et al. 2001). This does not imply thatall keratin K19⁺ cells are stem cells, since for instance the entirebasal layer of the skin is K19⁺ and this by far exceeds the expectednumber of stem cells. Therefore, in preferred embodiments, theimmortalised cell line comprises cells that are positive staining forthe keratin K19.

The most preferred embodiment of the present invention is a humansuprabasal-derived cell line possessing many of the characteristics of astem cells of the human breast gland and which is exemplified by theimmortalised D492 cell line. This immortalised cell line is deposited inaccordance with the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure at Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH (DSMZ) and has obtained the accession number DSMACC 2529. Other embodiments of the present invention is an immortalisedcell line that is capable of proliferating and capable ofdifferentiating into cells of mammary gland luminal epithelial andmyoepithelial cell lineages, and is derived from a cell selected fromthe group consisting of a rodent cell, a porcine cell, a ruminant cell,a bovine cell, a caprine cell, a equine cell, a canine cell, a ovinecell, a feline cell and a primate cell. In particular, an immortalisedcell line that is selected from the group consisting of cells from mice,rats and rabbits is an important embodiment of the present invention,since it opens for new experimental possibilities e.g. transplantationexperiments in syngeneic individuals, which is not practicable in thecase of human cells. However, an immortalised cell line that is a humancell line is a preferred embodiment.

The isolation and establishment of three immortalised cell lines (D492,D490 and TH69) and two non-immortalised suprabasal luminal epithelialcell cultures (TH82 and TH95) show that the method for isolation of anat least bi-potent mammary gland tissue cell which is described inexample 2 is a general method for the isolation of a bi-potent cell typesharing many of the characteristics of a stem cell of the breast gland.The method for isolation of an at least bi-potent mammary gland tissuecell, comprising the steps of: (i) separating of the breast tissue intotwo or more different cell types; (ii) culturing each of said differentcell types under cell differentiation conditions and; (iii) selectingthe cell type(s) that is/are capable of differentiating into at leasttwo morphologically and/or phenotypically different cell types. In onespecific embodiment of the method according to the invention, an atleast bi-potent cell which is derived from luminal epithelial cells of amammary gland and capable of proliferating and capable ofdifferentiating into cells of mammary gland luminal epithelial andmyoepithelial cell lineages is isolated.

One preferred method separating the breast tissue cells into two or moredifferent cell types is to disaggregate the tissue mechanically followedby an enzymatically disaggregation with collagenase, as described inexample 2.

Example 2 describes a preferred embodiment of the subsequent culture ofdisaggregated breast tissue. The culture is performed by placing theorganoids in collagen-coated culture flasks added CDM3 (Petersen and vanDeurs, 1987) culture medium, and kept 37° C. in a standard cellincubator in a humidified atmosphere of 75% N₂, 20% O₂, and 5% CO₂. Inthe experiment reported in example 2 the culture flasks were coated with8 μg/cm² collagen. While this collagen coating results in a surfacewhich is optimal for the plating of primary human breast epithelialcells, other coatings may be applied for non-human cells. For instancecoating with fetal calf serum has been reported to facilitate plating ofprimary epithelial cells. Also the exact amount of collagen may varysuch as between 0.1-100 μg/cm², 0.5-50 μg/cm², 1-30 μg/cm², 3-20 μg/cm²,5-15 μg/cm² or 6-10 μg/cm² and depend on the exact type and stock.Whereas the CDM3 medium is preferred it is contemplated that almostidentical cell cultures may be obtained with other media. One example isthe DMEM/F-12 medium 1:1 supplemented with 2 mM glutamine and a numberof growth factors, see table 3.

Both in the case of isolation of luminal epithelial cells andsuprabasal-derived epithelial cells the organoids was cultured with achange of medium three times a week. When the organoids had spread outto monolayers in primary culture cells were trypsinised and filtered asdescribed by (Péchoux et al. 1999) and all cell separations were carriedout by use of specific antibodies coupled to the matrix of the MiniMACSmagnetic cell separation system according to the manufacturer'sinstructions (Militenyi Biotech, Gladbach, GmBH). Within the concept ofthis invention is also the use of other systems for cell separationwhich is based on a stepwise selection or enrichment of cells whichexpress no or low levels of sialomucin and relative high levels ofepithelial specific antigen (ESA). One such cell separation method isflow cytometry (Stingl et al. 1998).

Isolating an at least bi-potent suprabasal positioned luminal epitheliacell of the breast by any such sequential procedure involving culture ofprimary mamma organoids followed by a immunological based enrichment ofESA⁺/MUC⁻ cells and resulting in a cell which is capable of forming acell culture comprising cells which are positive staining for theluminal epithelial marker ESA (ESA⁺) and negative or weakly positivestaining for sialomucin (MUC⁻), (ESA⁺/MUC⁻) cells are stipulated to bewithin the scope of present invention.

Such isolated cells have many important industrial uses. In oneembodiment of the invention the cells are used to establish a method fortesting the toxic effect, if any, of a substance on mammary glandepithelial cells. The method comprising: (i) culturing or maintainingthe cells in a non-toxic medium; (ii) adding the substance to be testedto the medium; and (iii) determining the response, if any, of the cells,including changes in cell growth rate, cell death rate, apoptosis, cellmetabolism, inter- as well as intra-cellular communication, adhesion,morphology, mRNA or protein expression and antigen expression.

The term “growth rate” is defined as the net increase in cell numberwithin a given time period.

The term “apoptosis” is used to describe the normal cellular processinvolving an active, genetically programmed series of events leading tothe death of a cell. Often toxic substances induce apoptosis in cells.In contrast to apoptosis, which is, a basic physiological processnecrosis describes the accidental cell death that is the cell's responseto a variety of harmful conditions and toxic substances.

The term “cell death rate” is defined as the number of cells dying perunit time. The cell death rate is determined by the rate of which cellsundergo apoptosis or necrosis.

The term “intercellular communication” is defined as signalling betweenindividual cells as elicited by cytokines, extracellular matrixcomponents, adhesive molecules or the like.

The term “intracellular communication” is defined as the signallingwithin an individual cell typically elicited by activation of membranebound or nuclear receptors or other cell signalling mediators.

The cells of the present invention are keratin K19-expressing,suprabasally located cells within the luminal epithelial lineage thatare putative precursor cells of terminal duct lobular units in the humanbreast. Since it long has been assumed that human breast canceroriginates from the luminal epithelial lineage within the terminal ductlobular units (TDLU), and since more than 90% of human breast carcinomasare keratin K19 positive, it appears that the cells of this inventionare identical to the cellular origin of most human breast cancers, andtherefore constitute a very attractive cellular model system for breastcancer. Thus an even more preferred embodiment of the invention is amethod for testing the carcinogenic effect, if any, of a substance onmammary gland epithelial cells, the method comprising: (i) culturing thecells of the invention in a growth medium which maintains the cells asnon-transformed cells; (ii) adding the agent, compound or factor undertest to the cell culture; and (iii) determining the neoplastic response,if any, of the so contacted cells by changes in morphology,tumorigenicity in animals, mRNA expression and/or antigen expression aswell as other changes associated with carcinogenesisi.

By the term “neoplastic response” the present inventors here refer totumor formation as a consequence of clonal expansion of geneticallyaltered cells.

By the term “tumorigenicity in animals” the present inventors here referto the important aspect of neoplastic cells to form tumours in animals.The present invention refers to a tumorigenicity test performed oneither syngeneic or immuno-incompetent animals. However in a preferredembodiment the tumorigenicity test comprise the introduction of saidtreated cells into an immune incompetent test animal.

By the term “carcinogenicy” the present inventors here refer to tumorformation related to the effect of tumor promoting or genotoxicexposures such as exposure to radiation, tar and various carcinogenicsubstances.

Since the cells of the invention are characterised by being at leastbi-potent cells with stem cell characteristics the cells can beexploited to test the ability, if any, of a substance to modulate thedifferentiation of non-terminal differentiated mammary gland epithelialcells. Thus one aspect of the invention relates to a method of testing asubstance for its ability to modulate the differentiation of mammarygland epithelial cells comprising: (i) culturing or maintaining thecells of the present invention in a medium which in itself does notmodulate the differentiation; (ii) adding the substance under test tothe cell culture; and (iii) determining the differentiation modulationresponses, if any, of the so contacted cells by changes in cell growthrate, cell death rate, apoptosis, cell metabolism, inter- as well asintra-cellular communication, morphology, mRNA or protein expression orantigen expression as well as other changes which is associated withdifferentiation.

An interesting aspect of the invention relates to the use of the cellsaccording to the invention to test substances for their ability tointeract with a particular cellular protein.

To ensure expression of the specific protein in question a nucleic acidfragment which includes a nucleic acid sequence encoding for thespecific protein may be obtained, inserted into a suitable expressionvector and the resulting “gene construct” transfected into any of thecells or cell lines of the present invention.

The term “expression vector” is used to denote a DNA molecule, linear orcircular, which comprises a segment encoding a polypeptide of interestoperably, linked to additional segments that provide for its expression.Such additional segments include promoter and terminator sequences, andmay also include one or more origins of replication, one or moreselectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both. Many such “expression vectors” aredescribed in the art. A comprehensive survey of such expression vectorscan be found in Ausubel et al. 2000, which is incorporated herein byreference.

The gene construct may be introduced into the host of this inventioncell by transduction or transfection. Detailed description of usefulmethods may be found in Ausubel et al. 2000, and Sambrook, et al. 1989,both of which is incorporated herein by reference.

The next step in the test is to add the substance to be tested to thecells; and finally to determine the interaction, if any, with thecellular protein. Such interaction may be deduced from changes in thephenotype, e.g. by changes in cell growth rate, cell death rate,apoptosis, cell metabolism, inter- as well as intra-cellularcommunication, morphology, mRNA or protein expression, antigenexpression or other changes which either directly or indirectly issupposed to be associated with said protein.

Since the growth of breast epithelial cells in vivo is guided byhormones such as estrogen and progesterone a particular importantvariation of this embodiment is a method for the detection ofinteraction between a cellular protein and a given substance in whichsaid cellular protein is selected from the group consisting of cellularreceptors in particular estrogen receptor-alpha, estrogen receptor-betaand progesterone receptor.

In the present context the term “receptor” denotes a cell-associatedprotein that binds to a bioactive molecule (i.e., a ligand) and mediatesthe effect of the ligand on the cell. Typically the binding of ligand toreceptor results in a conformational change in the receptor that causesan interaction between the effector domain and other molecule(s) in thecell. This interaction in turn leads to an alteration in the metabolismof the cell. Metabolic events that are linked to receptor-ligandinteractions include gene transcription, phosphorylation,dephosphorylation, increases in cyclic AMP production, mobilization ofcellular calcium, mobilization of membrane lipids, cell adhesion,hydrolysis of inositol lipids and hydrolysis of phospholipids. Ingeneral, receptors can be membrane bound, cytosolic or nuclear monomeric(e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor)or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor andIL-6 receptor).

One particular intriguing embodiment of the present invention is totransplant a vertebrate host with a cell according to present invention.Such cells could be transfected with a heterologous nucleotide sequenceand consequently express such heterologous protein. The term a“heterologous nucleotide sequence” is herein used to describe a DNAsequence inserted within or connected to another DNA sequence whichcodes for polypeptides not coded for in nature by the DNA sequence towhich it is joined. The term a “heterologous protein” is herein used todescribe a polypeptide that is being expressed in a context not found inthe non-transfected cells. Consequently, it is within the scope of thepresent invention to administer a given protein or gene of interest toan individual in need thereof, comprising the step of transfecting thecell-population of said at least bi-potent luminal derived epithelialcells of a mammary gland with a vector comprising DNA or RNA whichexpresses the protein or gene of interest and introducing thetransfected cell into said individual. Thus the use an such at leastbi-potent cell to prevent and/or treat cellular debilitations,derangements and/or dysfunctions and/or other disease states in mammals,comprising administering to a mammal a therapeutically effective amountof said cells, or cells or tissues derived therefrom is within the scopeof the present invention.

Since the at least bi-potent immortalised suprabasal-derived epithelialcell line of the present invention is capable of forming branchingstructures resembling terminal duct lobular units of the mammary glandnot only by marker expression but also in morphology it is contemplatedthat such cells can be used for tissue repair or transplantation. Forinstance if patients suffer from drastic mastectomies, it is possible toisolate at least bi-potent mammary gland tissue cells from their breastby the methods of the present invention. Further it is possible toimmortalise said cells, and use them for reimplantation to re-engineer abreast tissue in situ. In a further useful embodiment of the invention,a method of tissue repair or transplantation in mammals, comprisingadministering to a mammal a therapeutically effective amount of an atleast bi-potent luminal derived epithelial cells of a mammary gland, orcells or tissues derived therefrom is contemplated.

Adult stem-like cells of the breast could conceivably also be used forderivation of other tissues. While this technology has not yet beendemonstrated, the chances are that the present inventors may be able toderive both hair forming cells and skin tissues from these cells becausebreast cells share an embryonic origin with these cells.

In a still further aspect, the invention pertains to the use of said atleast bi-potent luminal derived epithelial cells of a mammary gland forthe formulation of a pharmaceutical composition comprising: atherapeutically effective amount of said cells, or cells or tissuesderived therefrom; and a pharmaceutically acceptable carrier. Tocomplement the pharmaceutical composition, said composition couldfurther comprise a proliferation factor or lineage commitment factor.

In a final aspect of the invention said at least bi-potent luminalderived epithelial cells of a mammary gland is used to produce adiagnostic agent comprising said cells, or any part thereof. One suchdiagnostic agent could be a specific antibody directed against anantigen specific for said at least bi-potent cell of a mammary gland.

Part of the invention described in the present application has, afterthe filing of the priority application of the present application, ledto the publication Gudjonsson, T., Villadsen, R., Nielsen, H. L.,Rønnov-Jessen, L., Bissell, M. J., Petersen, O. W. 2002. Isolation,immortalization, and characterization of a human breast epithelial cellline with stem cell properties. Genes Dev. 16:693-706 published on 15Mar. 2002.

Other embodiments of the invention will be apparent to those skilled inthe art from a consideration of the specification or practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope and spiritof the invention being indicated by the following claims.

LEGENDS TO FIGURES

FIG. 1. Identification of “suprabasal” luminal epithelial cells in thebreast.

-   A. Suprabasal cells belong to the luminal epithelial lineage. (a)    Double labelling immunofluorescence staining of epithelial specific    antigen (ESA) and sialomucin (MUC1). The arrow indicates an example    of an epithelial cell which apparently does not reach the lumen and    fails to express sialomucin. (b) Double labelling immunofluorescence    staining of ESA and α-smooth muscle actin. Note that the suprabasal    epithelial cells (arrow) are resting on a basal layer of α-smooth    muscle actin-positive myoepithelial cells. (x1100; bar=20 μm).-   B. A subset of cells within the luminal epithelial lineage is    sialomucin-negative. Uncultured, trypsinized breast epithelial cells    were double stained to demonstrate ESA (green) and sialomucin (red).    Whereas the majority of cells were double-stained for ESA and    sialomucin, a small fraction stained for ESA only (arrow). (x1100;    bar=20 μm).

FIG. 2. Isolation, immortalisation and characterization of luminal andsuprabasal-derived epithelial cells.

-   A. The luminal and suprabasal-derived epithelial cells differ by    sialomucin expression. Immunoperoxidase staining with ESA (upper    row) and MUC1 (lower row) of the luminal (left column) and the    suprabasal-derived epithelial cell line (right column). Note that    whereas the luminal epithelial-derived cell line is homogenous in    its staining pattern and expressed both ESA and sialomucin (MUC1),    the suprabasal epithelial cell line is heterogeneous (arrows) and    essentially negative for sialomucin. (x250; bar=50 μm).-   B. The cell lines express E6 and E7 stably. RT-PCR of HPV16 E6 and    E7 show that both the luminal and the suprabasal-derived epithelial    cells are stably transduced as compared to a negative control.-   C. The cell lines exhibit telomerase activity. TRAP assay of equal    numbers of luminal and suprabasal-derived epithelial cell lines    showed telomerase activity in transduced cells. Lane 1: molecular    weight markers, lane 2: luminal-derived epithelial cell line, lane    4: suprabasal-derived epithelial cell line, lane 3 and 5: heat    inactivated negative control of the cell lines, lane 6: positive    control pellet, lane 7: negative control without cell lysate, lane    8: positive control TSR8 control template.-   D. Both the luminal and suprabasal-derived epithelial cell lines    belong to the luminal epithelial lineage. Confluent cultures were    plated on Transwell filters and assayed for transepithelial    resistance (TER), and parallel monolayer cultures were    double-stained for claudin 1 as well as propidium iodide to    visualize the nuclei (inserts). Primary luminal epithelial cells    (LEP) and myoepithelial cells (MEP) are readily discriminated by TER    and claudin-i expression. The luminal and suprabasal-derived cell    lines resemble primary luminal epithelial cells by a high TER and a    prominent expression of claudin-1. (x400; bar=20 μm).

FIG. 3. Evidence for multipotency in the suprabasal-derived epithelialcell line.

-   A. Doublestaining with luminal epithelial K18 and myoepithelial K14    in clones of the luminal-derived cell line and the    suprabasal-derived cell line. Clonal cultures of the    luminal-derived (a) and the suprabasal-derived epithelial cells    (b, c) were double-stained with keratin K18 and K14. No evidence for    myoepithelial cells, i.e. no K14-staining was found in any of the    luminal-derived clones. Conversely, a mixture of K18- and    K14-positive cells was frequently present in the suprabasal-derived    epithelial clones. (x200; bar=40 μm).-   B. Evidence of spontaneous maturation to myoepithelial-restricted    cells. Immunoperoxidase staining of Thy-1, a marker for    myoepithelial cells, in cultures of suprabasal-derived cells    before (a) and after (b) purification in a Thy-1 retaining column.    The spontaneous occurrence of Thy-1 stained cells is limited to less    than 1% (arrows). However, upon purification, a myoepithelial    restricted subline can be obtained which also express α-smooth    muscle actin as shown by RT-PCR(c). (x250; bar=50 μm).-   C. Evidence for maturation to luminal epithelial-restricted cells.    Suprabasal-derived epithelial cells were cleared of    sialomucin-positive cells and stained for sialomucin after 2 weeks    (a, arrows), and after further retention of the newly formed    sialomucin-positive cells (b). The MUC stainings was confirmed by    RT-PCR (c). (x250; bar=50 μm).

FIG. 4. Only suprabasal-derived epithelial cells give rise to terminalduct lobular units (TDLU).

-   A. Luminal epithelial- and myoepithelial-derived cells make colonies    with distinct morphologies in a laminin-rich gel. Immortalized    (a, c) and primary (b, d) luminal epithelial-derived cells (a, b)    and myoepithelial-derived cells (c, d) were embedded as single cells    in a laminin rich gel. Both immortal luminal epithelial- and    myoepithelial-derived cells resembled the corresponding primary    cells. Whereas the luminal epithelial cells formed acinus-like    spheres with a central lumen, the myoepithelial cells formed    irregular solid clusters of cells. (x200; bar=25 μm)-   B. Suprabasal-derived epithelial cells make an elaborate TDLU-like    structure in a laminin-rich gel. Suprabasal-derived epithelial    cells (a) were embedded as single cells in a laminin-rich gel and    compared with the morphology of freshly isolated, uncultured TDLU    organoids (b). Both consist of small branching ductules terminating    in globular acinus-like structures. (x100; bar=50 μm)-   C. Quantitation of TDLU-like structures in laminin-rich gels.    Luminal and suprabasal-derived epithelial cells and    myoepithelial-derived cells were embedded inside laminin-rich gels    and allowed to grow for 12 days. The number of TDLU-like structures    was quantified by phase contrast microscopy.-   D. Only suprabasal-derived epithelial colonies in laminin-rich gels    resemble TDLU in vivo. Sections of laminin-rich gels containing    luminal-derived (left column) and basal-derived epithelial cells    (middle column) were compared with sections of normal breast tissue    (right column) and doublestained for ESA and keratin K14 (a-c), and    propidium iodide and laminin-1 (d-f). Only the suprabasal-derived    epithelial cells showed a differentiation pattern reminiscent of    normal breast tissue with an inner layer of ESA-positive cells and    an outer layer of K14-positive/ESA-negative cells and basal    deposition of a laminin-1 containing basement membrane (x400;    bar=151 μm).

FIG. 5. The suprabasal-derived cells are keratin K19-positive similar toa subpopulation of cells in TDLU and neoplastic breast epithelial cellsin vivo.

-   A. Luminal epithelial- and suprabasal-derived epithelial cells    differ by expression of mRNA for keratin K19. RT-PCR of keratin K19    in luminal epithelial- and suprabasal-derived epithelial cells with    expression in the suprabasal-derived cells only.-   B. Luminal epithelial- and suprabasal-derived epithelial cells    differ by expression of keratin K19. Immunoblot of keratin K19 of    protein lysates from luminal epithelial- and suprabasal-epithelial    derived cells showing expression in the suprabasal-derived cells    only.-   C. Keratin K19 staining in cultures of luminal- and    suprabasal-derived epithelial cells. Cultures were stained for    keratin K19 by immunoperoxidase and counterstained with hematoxylin.    Whereas the luminal-derived epithelial cells were completely    negative, the other cell line was heterogeneous with a large    contribution from keratin K19-positive cells. (x250; bar=50 μm).

FIG. 6. Keratin K19 staining in TDLU and infiltrating ductal carcinoma(IDC).

-   -   Section of breast tissue showing TDLU (a) and IDC (b) stained        for keratin K19 and counterstained with hematoxylin. Note the        heterogeneous staining and the presence of several stained        suprabasal cells in the TDLU and the more widespread staining of        neoplastic epithelial cells in the carcinoma. (x250; bar=50 μm).

FIG. 7. Clonal segregation of keratin K19-positive and K14-positivecells in two- and three-dimensional culture and mouse implants ofsuprabasal-derived cells.

-   -   Clonal culture of suprabasal-derived cells on monolayer        collagen-coated plastic (A), in a laminin-rich gel (B) and        implanted orthotopically in the nude mouse (C) double-labelled        with keratin K19 and keratin K14. The monolayer cultures show        distinct evidence of bi-potency, and in three-dimension in        culture as well as in vivo this organizes further into TDLU-like        structures including terminal ducts and acini (x400; 40 μm).

EXAMPLES Example 1

Identification of “Suprabasal” Luminal Epithelial Cells in the Breast

In culture, a putative “stem” cell of the human breast was defined basedon a positive staining for the luminal epithelial marker ESA and anegative or weakly positive staining for sialomucin (MUC) (Stingl et al.1998). To investigate if such a candidate stem cell could be identifiedin human breast in vivo, the present inventors double-stainedhistological sections of normal human breast tissue withepithelial-specific antigen (ESA) and sialomucin (MUC).

In general, immunocytochemistry and confocal microscopy was performed asfollows. Normal human breast tissue was obtained as biopsies frompatients undergoing reduction mammoplasty for cosmetic reasons. The useof human material has been reviewed by the Regional Scientific-EthicalCommittees for Copenhagen and Frederiksberg, Denmark and approved withreference to (KF) 01-161/98. The tissue was frozen in n-hexan (Merck,Darmstadt, Germany) and mounted in Tissue Freezing Medium™ (LeicaInstruments, Heidelberg, GmbH) for sectioning. Frozen tissue wassectioned at an 8-μm setting in a cryostat. The sections and cellcultures were dried for 15 min at room temperature and fixed in methanolas previously described (Petersen and van Deurs 1988). Primaryantibodies directed against sialomucin (MAM6, clone 115D8, BiogenesisLtd., Poole, UK), epithelial-specific antigen (ESA; VU-1D9, NovoCastra,Newcastle upon Tyne, UK ) and α-smooth muscle actin (1A4, Sigma-Aldrich,Vallensbœk, Denmark) were used. For an overview of the primaryantibodies used throughout the study, see Table 4. Rabbit anti-mouseimmunoglobulins (Z259, DAKO, Glostrup, Denmark) were used as secondaryantibodies and a peroxidase conjugated anti-peroxidase mouse mAb wasused as tertiary antibody (P850, DAKO, Glostrup, Denmark). Theperoxidase reactions were performed using 0.5 mg/ml 3,3-diaminobenzidine(Sigma-Aldrich, Vallensbœk, Denmark) and 0.5 μl/ml of 30% H₂O₂ (Merck,Darmstadt, Germany, purchased from Struers KEBO LAb A/S, Albertslund,Denmark) for 10 minutes. The sections were counterstained withhematoxylin (Mayer's hematoxylin, MHS-16, Sigma-Aldrich, Vallensbœk,Denmark). For double-labeling experiments the present inventors usediso-type specific antibodies, all from Southern Biotechnology (SouthernBiotechnology Associates, Birmingham, Ala.) as previously described(Rønnov-Jessen et al. 1995). Antibody incubations were carried out for30 min, and specimens were rinsed twice for 5 min each, all at roomtemperature. Some sections received a nuclear counter stain with 1 μg/mlpropidium iodide (Molecular Probes, Eugene, Oreg.). Afterwards sectionswere mounted with coverslips by use of Fluoromount-G (SouthernBiotechnology Associates, Birmingham, Ala.) supplemented with 2.5 mg/mln-propyl gallate (Sigma-Aldrich, Vallensbœk, Denmark) as previouslydescribed (Rønnov-Jessen et al. 1992). Immunofluorescence was visualizedusing a Zeiss LSM 510 laser scanning microscope (Carl Zeiss, Jena,GmbH). Sections were observed by use of a 20×, 40×, 63× or 100×objective and sliced in the z-plane into 0.25 μm-thick focal planes andexposed to visualize FITC and Texas red or propidium iodide.

Most luminal epithelial cells stained as expected, i.e. MUC wasexpressed on the apical surface and ESA at the basolateral surface (FIG.1A).

However, it was also evident that a subset of ESA⁺ cells in occasionalacinar profiles was indeed abluminal in location with no visibleextensions reaching the lumen (arrow, FIG. 1A, a). To confirm that thesecells were distinct from myoepithelial cells, the present inventorsdouble-stained for ESA and the myoepithelial marker α-smooth muscleactin (α-sm actin). As expected, myoepithelial cells were negative forESA, while a very minor population of basal cells were suprabasal anddid not appear to reach the lumen or stain with α-sm actin (arrow, FIG.1A, b). If these cells truly never reached the lumen—not even outsidethe sectioned plane—a sample of smeared trypsinized, uncultured breastcells should contain two ESA⁺ luminal epithelial populations: one majorbeing MUC⁺ and a minor being MUC⁻. This was found to be the case asevidenced by doublestaining of such smears for ESA and MUC (FIG. 1B, aand b). The average frequency of MUC⁻ cells in such preparations was8±3%.

From these experiments it is concluded that suprabasally positioned,abluminal cells within the luminal epithelial lineage also exist in thehuman breast.

Example 2

Isolation, Immortalization and Characterization of Luminal andSuprabasal-Derived Epithelial Cells.

In order to show that the cells described in example 1 indeed have stemcell properties, the cells were isolated by immunomagnetic sorting andcharacterized.

Briefly, the luminal epithelial cells were purified from two consecutivesialomucin-columns and the suprabasal epithelial cells were purified asthe flow-through from a sialomucin-column which was later retained in anESA-column. To generate cell lines, the present inventors immortalisedboth populations with an E6/E7 construct of HPV16. The resultingestablished cell lines are referred to below as the luminal andsuprabasal-derived epithelial cells, respectively.

Cell Culture

Breast luminal epithelial cells were generated from primary cultures ofbiopsies from patients undergoing reduction mammoplasty for cosmeticreasons. The tissue was prepared as previously described (Péchoux et al.1999). Briefly, it was mechanically disaggregated followed by enzymaticdisaggregation with collagenase (CLSIII, 900 units/ml, Worthington,purchased from Medinova, Hellerup, Denmark) to release epithelialorganoids. The organoids were plated in CDM3 medium (Petersen and vanDeurs 1987) on collagen-coated (8 μg/cm²; Vitrogen-100, Cohesion, PaloAlto, Calif.) T-25 flasks (Nunc, Roskilde, Denmark). Cells were kept at37° C. in a Heraeus incubator in a humidified atmosphere of 75% N₂, 20%O₂, and 5% CO₂, and the medium was changed three times a week. In someinstances organoids were trypsinized directly after collagenasedigestion to obtain uncultured single cells for smears, which were fixedin methanol (Merck, Darmstadt, Germany) and further analysed.

Luminal Cells

Luminal epithelial cells were purified after organoids had spread out tomonolayers in primary culture. Cells were trypsinized and filtered aspreviously described (Péchoux et al. 1999). All cell separations werecarried out by use of the MiniMACS magnetic cell separation systemaccording to the manufacturer's instructions (Militenyi Biotech,Gladbach, GmBH). The luminal epithelial cells were separatedimmunomagnetically from myoepithelial cells by retention in twoconsecutive anti-sialomucin (MAM6, clone 115D8, Biogenesis Ltd., Poole,UK) columns, and plated in CDM6 as peviously described (Péchoux et al.1999). The cells were immortalised in passage 3 (for procedure, pleasesee below), and cultured in CDM3 (Petersen and van Deurs 1987) untilpassage 11, where the medium was switched to H14 (Blaschke et al.1994)(D382, Table III).

Suprabasal-Derived Epithelial Cells

Similar to the situation with the luminal epithelial cellssuprabasal-derived epithelial cells were purified after organoids hadspread out to monolayers in primary culture. Cells were similarlytrypsinized and filtered and all cell separations were carried out byuse of the MiniMACS magnetic cell separation system according to themanufacturer's instructions (Militenyi Biotech, Gladbach, GmBH).

The luminal cell population containing the suprabasal ESA⁺/MUC⁻ cellswas isolated as the flow-through of an anti-sialomucin (MAM6, clone115D8, Biogenesis Ltd., Poole, UK) column, and plated in Dulbecco'sModified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12, 1:1, GibcoBRL,LifeTechnologies, Tåstrup, Denmark) with glutamine (2 mM, GibcoBRL,LifeTechnologies, Tåstrup, Denmark) supplemented with cholera toxin (10ng/ml, Sigma-Aldrich, Vallensbœk, Denmark), epidermal growth factor (100ng/ml, PeproTech EC LTD, London, UK) and keratinocyte growth factor (10μg/ml, PeproTech EC LTD, London, UK). Since all cultured breastepithelial cells in contrast to fibroblasts expressed β4-integrin(Gudjonsson et al. 2002a), residual fibroblasts were removed byretaining epithelial cells in a β4-integrin (3E1, ChemiconInternational, Temecula, Calif.) column. The purified epithelial cells,now in third passage, were then plated in DMEM/F-12 (1:1, GibcoBRL,LifeTechnologies, Tåstrup, Denmark) supplemented with glutamine (2 mM,GibcoBRL, LifeTechnologies, Tåstrup, Denmark), 10% fetal calf serum(E.C. approved, virus and mycoplasma tested, GibcoBRL, LifeTechnologies,Tåstrup, Denmark), insulin (3 μg/ml, Boehringer Mannheim, Roche,Hvidovre, Denmark), hydrocortisone (1.4×10⁶ M, Sigma-Aldrich,Vallensbœk, Denmark) and epidermal growth factor (100 ng/ml, PeproTechEC LTD, London, UK), and immortalised (please see below). In passage 6,the medium was switched to H14 (Blaschke et al. 1994).

D492

The suprabasal epithelial cells (D492, DMSZ no. DMS ACC 2529) werecollected in passage 27 by retention of cells by an anti-ESA (VU-1D9,NovoCastra, Newcastle upon Tyne, UK) column (D492, Table III).

D490

The procedure was repeated with luminal cells from another biopsy withthe following modifications: retention of β4-positive epithelial cellsin second passage, flow-through of an anti-sialomucin column in passage3, followed by immortalisation, switch to H14 in passage 6, andisolation of suprabasal epithelial cells, comparable to D492, byretention in an anti-ESA column in passage 10 (D490, Table III).

TH69

Finally, a third suprabasal cell line (TH69, Table III) was generated asthe flow-through of an anti-sialomucin column, followed by retention inan anti-ESA column in second passage, immortalisation in passage 3, andsubsequent switch to H14 in passage 6.

Non-Immortalised Suprabasal-Derived Epithelial Cells

To further verify that suprabasal cells with similar properties could beobtained without immortalisation, ESA⁺/MUC⁻ suprabasal cells werepurified directly from primary cultures from two different biopsies asthe flow-through of an anti-sialomucin (MAM6, clone 115D8, BiogenesisLtd., Poole, UK) column followed by retention in an anti-ESA (VU-1D9,NovoCastra, Newcastle upon Tyne, UK) column (TH82 and TH95, Table III).The purified third passage suprabasal cells were embedded directly in300 μl Matrigel® and cultured for 2 weeks in CDM3 medium (Petersen andvan Deurs 1987), and within this period TDLU-like structures wereformed.

Myoepithelial-Derived Cells

For isolation and purification of myoepithelial-derived cells thepresent inventors used an antibody against Thy-1 (AS02, Dianova,Hamburg, GmbH) (Gudjonsson et al. 2002a and b).

Establishment of Immortalized Cell Lines and Clonal Cultures

The luminal epithelial and suprabasal epithelial cells were transducedwith sterile filtered retrovirus-containing supernatant from the PA317LXSN HPV16E6E7 packaging cell line (CRL-2203, ATCC, Rockville, Md.) inthe presence of 8 μg/ml polybrene (Sigma-Aldrich, Vallensbœk, Denmark)(Wazer et al. 1995). Transduced cells were selected in the presence of100 μg/ml G418 (Life Technologies, Tåstrup, Denmark). Established celllines were kept routinely in H14 medium (Blaschke et al. 1994). Clonalcultures were prepared according to a protocol for prostate epithelialcells (Hudson et al. 2000). Briefly, 1000 or 5000 cells (10 times morein 3-dimensional laminin rich gels) were plated onto collagen-coated(Vitrogen-100, Cohesion, Palo Alto, Calif.) T-25 flasks (Nunc, Roskilde,Denmark) in serum-free H14 medium and kept for 2 weeks prior tostaining.

The resulting immortalised cell lines were tested for their tumorigenicproperties in a standard tumorigenicity assay. Briefly, 10⁷ cellsimmortalised cells were inoculated subcutaneously into BALB/C nude mice.The mouse were followed by weekly inspections by palpation of theinjection site. None of the cell lines were tumorigenic even afterprolonged incubation.

Telomerase-Activity

The telomerase activity was determined with the TRAP assay using theTRAPeze Telomerase Detection Kit (Intergen, Oxford, UK) according to themanufacturer's instructions. Cells were grown to 70-80% confluence,trypsinized and counted. A lysate volume equal to 1000 cells was usedfor each reaction, and electrophoresed on a 12% nondenaturing acrylamidegel (BioRad, Herlev, Denmark), stained in SYBR green 1 (MolecularProbes, Leiden, The Netherlands) and visualized by UV transilluminationand image recording in a Gel Doc 1000 (BioRad, Herlev, Denmark).

Transepithelial Electrical Resistance (TER)

For TER measurements, cells were plated on polycarbonate filters with apore size of 0.41 μm (Corning Costar Corporation, Cambridge, Mass.) andallowed to reach confluency. A Millicell-ERS volt-ohm meter (Millipore,Hedehusene, Denmark) was used to determine the TER value. All TER valueswere normalized for the area of the filter and were obtained afterbackground subtraction. All experiments were done in triplicate.

Results

As seen in FIG. 2A, the resulting established cell lines were ESA⁺/MUC⁺(D382) and ESA⁺/MUC⁻ (D492), respectively. The cell lines displayedimmortalised characteristics: they have been cultured for more than 50passages over 2 years and continue to express both E6 and E7 (FIG. 2B),and a distinct telomerase activity which is absent from finite life spanbreast epithelial cells (FIG. 2C) (Stampfer et al 2001). Importantly,the immortalised D492 cell line were tested and found non-tumorigenicand in the case of the D492 cell line it was found that it has a diploidkaryotype (46, XX) even after more than one year in culture andpassages.

Three suprabasal cell lines with similar characteristics wereestablished (D492, D490 and TH69). The latter was isolated as theflow-through of an anti-sialomucin column, directly followed byretention in an anti-ESA-column prior to immortalisation. Moreover, theretention step in a β4-integrin column was omitted. This indicates thatthe ESA⁺/MUC⁻ cells are indeed present initially and thus, do not occuras a result of immortalisation.

Whereas the luminal-derived epithelial cell line continued to behomogeneous (FIG. 2A, left), the suprabasal-derived epithelial cell linecontained occasional subpopulations of ESA⁻ cells and MUC⁺ cells (FIG.2A, right, arrow). Double immunofluorescence staining for ESA⁻ and MUC(not shown) revealed that this cell line contained three cellularsubtypes: the majority population was ESA⁺/MUC⁻ and two minorpopulations were either ESA⁻/MUC⁻ or ESA⁺/MUC⁺. To substantiate thatboth cell lines belonged to the luminal epithelial lineage, even thoughone of them was essentially devoid of luminal epithelial MUC expression,the present inventors tested for a marker that is a hallmark ofglandular epithelial phenotype—that of functional tight junctions. Thiswas carried out by staining for the tight junction proteins claudin andoccludin as described in example 1 but using antibodies against occludin(OC-3F10, Zymed Laboratories, San Francisco, Calif.) and polyclonalclaudin-1 (Zymed Laboratories, San Francisco, Calif.) as primaryantibodies, see also Table 4. Furthermore the level of transepithelialelectrical resistance (TER) in confluent cultures on transwell filterswas measured. By these criteria, primary luminal epithelial cells werereadily distinguished from primary myoepithelial cells (FIG. 2D). Asalso seen in FIG. 2D, the newly established cell lines both resembledluminal epithelial cells by staining at the cell boundaries for tightjunction proteins (see inserts) and exhibiting a high TER. The presentinventors conclude that both cell lines belong to the luminal epitheliallineage.

Example 3

Clonal Cell Lines of the Suprabasal-Derived Epithelial Cell Line areMultipotent

Clonal cultures were established and double-stained for keratin K18(luminal marker) and K14 (myoepithelial marker) as described in example1 by using antibodies against keratin K18 (F3006; Trichem Aps, Denmark)and keratin K14 (LL002, NovoCastra, Newcastle upon Tyne, UK) as primaryantibodies, se also Table 4.

Whereas the luminal-derived epithelial cell line did not generate anymyoepithelial cells and stained for K18 only, the suprabasal-derivedcell line readily formed mixed clones of luminal epithelial andmyoepithelial cells (FIG. 3A). The present inventors found that thesemyoepithelial cells represented a primitive level of myoepithelialdifferentiation because <1% of the cells expressed other myoepithelialmarkers such as Thy-1 (FIG. 3B, a). The staining for Thy-1 was performedas described in example 1 using the Thy-1 (AS0-2, Dianova, Hamburg,GmbH) as primary antibody, see also Table 4. However, if the cells wereretained in a Thy-1 column, a myoepithelial-restricted subline could begenerated which also expressed α-smooth muscle actin which is restrictedto postmitotic myoepithelial cells in vivo (FIG. 3B). The staining forα-smooth muscle actin were performed as described in example 1. Thepresent inventors reasoned that, if K18⁺ cells were also precursor cellsof a lineage-restricted progeny within the luminal compartment, theycould further mature within this compartment to differentiated cells.Sialomucin-expressing cells were eliminated by retention on asialomucin-retaining column, but evidence for spontaneous maturationinto sialomucin-positive cells was provided by the reccurrence of thesecells within 2 weeks (FIG. 3C, a). These cells in turn could be retainedin a similar column and kept as lineage-restricted in high-densitycultures in the presence of serum (FIG. 3C, b, c). Taken together, theseobservations provide evidence for the existence of a suprabasal,multipotent epithelial cell belonging to the luminal epithelial lineagethat can give rise to differentiated myoepithelial and luminalepithelial cells, and their precursors.

Example 4

Only Suprabasal-Derived Epithelial Cells Give Rise to Terminal DuctLobular Units (TDLU)

In the mouse and rat, the standard criteria for the presence of stemcells has been the ability to regenerate the entire structure of themammary gland upon reimplantation of cells in syngeneic gland-free fatpads (Smith and Medina 1988). The present inventors performed a similartest in a three-dimensional laminin-rich gel as previously described(Petersen et al. 1992). Briefly, for three-dimensional cultures, 2.5×10⁵luminal-derived, suprabasal-derived, and myoepithelial-derived celllines were plated separately inside rBM (Matrigel®, lot# 40230A, BectonDickinson, Mass.). Experiments were carried out in 24 well dishes (Nunc,Roskilde, Denmark) using 300 μl Matrigel in which single cells weresuspended. Primary luminal epithelial cells, myoepithelial cells anduncultured terminal duct lobular unit (TDLU) organoids from the breastwere used as control (Petersen et al. 1992). The percentage of TDLUformation defined by branching of cell clusters was quantified by phasecontrast microscopy using a 10× objective and a 10× eye piece.

In this test the luminal-derived epithelial cell line or themyoepithelial-derived cells yielded morphologies very similar to whathas already been described for primary breast cells (Petersen et al.1992) (FIG. 4A). In contrast, embedding the suprabasal-derivedepithelial cells gave rise to formation of larger, more elaboratemorphologies resembling the entire functional unit of the breast gland,i.e. the terminal duct lobular unit (TDLU) (FIG. 4B and C). A similarfrequency of TDLU structures (73%) was recorded if suprabasal cells werefreshly prepared from primary cultures from two different biopsies.Sections of laminin-rich gels embedded with the suprabasal-derivedepithelial cells and stained for differentiation markers of normalbreast revealed a remarkable similarity to TDLU 's in vivo (FIG. 4D).

To provide evidence that this ability to generate a suprabasal-derivedepithelial cell line from the human breast with all the above mentionedstem cell characteristics was not simply of this particular cellisolation the present inventors repeated the entire protocol includingimmortalisation of cells from a different biopsy twice, and the presentinventors were able to reproduce the TDLU assay. Collectively, thesedata are in strong support of “suprabasal” cells within the luminalepithelial lineage as precursors of the TDLU, the functional units inthe human breast.

Example 5

The Suprabasal-Derived Cells are Keratin K19⁺ Similar to a Subpopulationof Cells in TDLU and Neoplastic Breast Epithelial Cells in vivo

To identify a candidate subpopulation within TDLU in which the stem cellcould reside, the present inventors performed an analysis of the luminalepithelial markers expressed by the two established cell lines.

Cells were stained for keratin K19 as described in example 1 but usingkeratin K19 (BA17 and RCK108, DAKO, Glostrup, Denmark) as primaryantibody, see also Table 4. Thus, keratin K19 was identified as adistinctive trait expressed only by the suprabasal-derived epithelialcells (Table 1). This difference in phenotype was confirmed further byreverse transcription PCR (RT-PCR), immunoblotting and immunostaining(FIG. 5A-C).

RNA isolation and reverse transcription PCR were performed as follows:Total RNA was extracted from monolayer cultures with Trizol® accordingto the manufacturer's instructions (Life Technologies, Tåstrup,Denmark). DNase-treated (DNase I Amp Grade, Life Technologies, Tåstrup,Denmark) total RNA (1.3 μg) was used as template for first strandsynthesis with oligo dT primers (SuperScript First-Strand SynthesisSystem for RT-PCR, Life Technologies, Tåstrup, Denmark) in a 20 μlvolume. A volume of 1 μl from this cDNA served as template for thesubsequent PCR-amplifications in a PE 9700 thermal cycler with a heatedlid (Applied Biosystems, Nœrum, Denmark), using primers (purchased fromTAG Copenhagen, Copenhagen, Denmark) specific for HPV16 E6 and E7 (HPV16E6 and HPV16 E7, respectively), keratin K19 (K19), sialomucin (MUC1),α-smooth muscle actin (αSM Actin) and glyceraldehyde 3-phosphatedehydrogenase (GAPDH). The primer sequences, annealing temperature(T_(A)) and number of amplification cycles for each reaction, as well asthe resulting product size are listed below (Table 2). Each PCR-reactionwas initiated with a 15 min incubation step at 95° C., followed by thespecified number of cycles with denaturation at 94° C., annealing at thespecified T_(A), and extension at 72° C., for 1 min each, followed by afinal extension step at 72° C. for 7 min. Each reaction was performed ina 50 μl volume containing 2.5U HotStar taq polymerase (Qiagen, KEBO LabA/S, Albertslund, Denmark), 10×PCR buffer including MgCl₂ (Qiagen, KEBOLab A/S, Albertslund, Denmark), 200 μM dNTP (Roche, Hvidovre, Denmark)and 200 nM of forward and reverse primers.

Control amplification was performed on RNA samples not subjected toreverse transcription to verify that no contaminating genomic DNA waspresent (data not shown). The PCR products were analysed byelectrophoresis in 1.5% agarose gels (GibcoBRL, Life Technologies,Tåstrup, Denmark).

Brieflly, the immunoblotting was performed by lysing semi-confluent T-25flasks of luminal- and suprabasal-derived epithelial cells, and T47Dbreast cancer cells (positive control;) for 30 min at 4° C. in buffercontaining 1% Triton X-100 (Merck, Darmstadt, Germany), 1% deoxycholicacid (Sigma-Aldrich, Vallensbœk, Denmark), 10% glycerol (ApotekAusturbœjar, Reykjvik, Iceland), 20 mM Tris-HCl (USB, Cleveland, Ohio,US), pH 7.5,150 mM NaCl (Merck, Darmstadt, Germany), 2.5 mM EDTA(Titriplex II, Merck, Darmstadt, Germany), 1 mMPMSF(phenylmethylsulfonyl flouride, Sigma-Aldrich, Vallensbœk, Denmark),1% aprotinin (trasylol, Sigma-Aldrich, Vallensbœk, Denmark), 100 μMNaVO₃ (Sigma-Aldrich, Vallensbœk, Denmark). The lysates were centrifugedand samples were subjected to 12% SDS-PAGE and run at 35 mV for 4 hours.The loading of lanes was equilibrated based on protein determinations bythe Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, Calif.). Thesamples were electrophoretically transferred to Immun-Blot PVDF Membrane(BioRad Laboratories, Hercules, Calif.) at 400 mA for 3-4 h at 4° C. in20% methanol (Merck, Darmstadt, Germany), 0.2 M glycine (USB, Cleveland,Ohio, US), and 25 mM Tris-HCl (USB, Cleveland, Ohio, US). Blots wereblocked in phosphate-buffered saline containing 5% dried milk (Osta ogSmjörsalan, Reykjavik, Iceland) and 0.05% Tween-20 (USB, Cleveland,Ohio, US) for 1 h at room temperature before probing with anti-keratinK19. The blots were washed three times for 10 min in phosphate-bufferedsaline containing 0.05% Tween-20 (USB, Cleveland, Ohio, US) and thenincubated with the anti-mouse IgG, horseradish peroxidase linked wholeantibody (NA931, Amersham Pharmacia Biotech, Amersham, UK). Afterwashing, bound antibodies were visualized using the ECL immunoblottingdetection system (Amersham Pharmacia Biotech, Amersham, UK).

Immunostaining was preformed as described previously. Primary antibodiesdirected against keratin K19 (BA17 and RCK108, DAKO, Glostrup, Denmark)were used, see also Table 4.

In tissue sections of normal breast the present inventors found alimited expression of keratin K19 including staining of suprabasal cells(FIG. 6 a, arrow), whereas in the majority of breast carcinomas, theneoplastic epithelial cells stain positive for keratin K19 (FIG. 6 b).If the keratin K19⁺ cells were indeed potential stem cells, then thesuprabasal-derived epithelial cell line should show evidence ofmultipotency with regard to keratin K19 expression. As seen in FIG. 7A,clones could be identified which diversified into both K14⁺ and K19⁺cells. Similarly, cloning in laminin-rich gels also resulted information of TDLU structures, which showed correct segregation of cellsinto suprabasally/luminally positioned K19⁺ cells and basally locatedK14⁺ cells (FIG. 7B).

Finally, to provide yet further evidence for the morphogenic potentialof these cells in vivo, the present inventors inoculated the fat pad ofnude mice after preembedding the cells in a mixture of collagen gel andrBM (Yang et al. 1994). Briefly, luminal- and suprabasal-derivedepithelial cells were inoculated subcutaneously into BALB/C nude miceafter preembedding 106 cells in 500 μl of a mixture of collagen and rBM(20% Matrigel® (lot# 40230A, Becton Dickinson, Mass.)/80% collagen(Vitrogen-100, Cohesion, Palo Alto, Calif.)) (Yang et al. 1994). Themice were sacrificed after one week and the implants were sectioned andstained (see above).

The present inventors used this assay to show that thesuprabasal-derived epithelial cell line segregated intosuprabasal/luminal keratin K19⁺ cells and basal keratin K14⁺ cells (FIG.7C).

It is concluded that the suprabasal-derived epithelial cells have aneasily identifiable equivalent in vivo which appears to be identical tothe stem cell of normal human breast.

The experiments described here establish both the existence of candidatemultipotent stem cells in the human breast and the fact that they can beimmortalised without loss of stem cell potential. The experiments alsooutline a method for their isolation and further characterisation. Themultipotent cell line was derived from a suprabasally located cell invivo which nevertheless belongs to the luminal epithelial lineage asevidenced by expression of ESA, claudin-1, keratins K18 and K19, butalso by the ability to form monolayers that display a high TER.Embedding clonal populations in a three dimensional basement membranegel or in mammary fat pads of mice reveal that the suprabasal-derivedcell line recapitulates an elaborate morphology closely reminiscent ofTDLU in vivo. The present inventors propose that cells with a suprabasalposition within the luminal epithelial lineage (K19⁺, ESA⁺, MUC⁻) arecandidate breast stem cells and putative precursors to human breastTDLUs. TABLE 1 Keratin K19 is a distinctive trait expressed only bysuprabasal-derived epithelial cells as revealed by immunocytochemicalstaining. Luminal-derived Suprabasal-derived epithelial Differentiatedtrait epithelial cells cells Claudin-1 + + Occludin + +epithelial-specific + + antigen (ESA) Keratin K18 + + Keratin K19 ÷ +sialomucin (MUC) + ÷/+ E-cadherin + +See table 4 for description of primary antibodies.

TABLE 2 Primer sequences Amplification Product Size Primer Sequence(5′-3′ direction) T_(A) (C) cycles (bp) HPV16 E6-FW GCAACAGTTACTGCGACGTG55° 30 234 HPV16 E6-RV GGACACAGTGGCTTTTGACA HPV16 E7-FWGATGGTCCAGCTGGACAAGC 55° 30 143 HPV16 E7-RV GTGCCCATTAACAGGTCTTC K19-FWGAGGTGGATTCCGCTCCGGGCA 58° 25 462 K19-RV ATCTTCCTGTCCCTCGAGCAG MUC1-FWGTACCATCAATGTCCACGAC 60° 30 351 MUC1-RV CTACGATCGGTACTGCTAGG αSMActin-FW GGAATCCTGTGAAGCAGCTC 56° 32 1200  αSM Actin-RVCACAGTTGTGTGCTAGAGACAGAG GAPDH-FW GAAGGTGAAGGTCGGAGT 54° 25 226 GAPDH-RVGAAGATGGTGATGGGATTTC

Primers were specific for human papilloma virus 16 E6 and E7 (HPV16 E6and HPV16 E7, respectively), keratin K19 (K19), Sialomucin (MUC1), αsmooth muscle actin (αSM Actin) and glyceraldehyde 3-phosphatedehydrogenase (GAPDH). FW: forward primer; RV: reverse primer. TABLE 3Biopsy P595 (D492, suprabasal cell line) Exp no. Passage MACS MediaImmortalized D470 1 CDM3 D480 2 MAM6− § + CT + EGF + KGF D486 3 β4+ § +10% FCS + I + H + EGF D492 3 § + 10% FCS + X I + H + EGF 6 H14 27  ESA+H14 Biopsy P594 (D490, suprabasal cell line) Exp. no Passage MACS MediaImmortalized D467/1 1 CDM3 D467/8 1 § + 10% FCS + I + H + EGF D467/6-7 1§ + CT + EGF 475/2 2 MAM6− § + 10% FCS + I + H + EGF 477/1 2 β4+ § + 10%FCS + I + H + EGF 478/1 2 β4+ § + CT + EGF D490/1 3 MAM6− § + 10% FCS +I + H + EGF D500(D490/1) 3 § + 10% FCS + X I + H + EGF 6 H14 10  ESA+H14 Biopsy A245 (TH69, suprabasal cell line) Exp. no Passage MACS MediaImmortalized A245 1 CDM3 TH65 2 MAM6− CDM3 TH68 3 ESA+ CDM3 TH69 3 CDM3X 3 CDM3 + 10% FCS 4 CDM3 6 H14 Biopsy P591 (D382, luminal cell line)Exp no. Passage MACS Media Immortalized D346 1 CDM3 D360 2 MAM6+ CDM6D369 3 MAM6+ CDM6 D382 3 CDM3 X 11  H14 Biopsy A253 (TH82, primarysuprabasal cells) Exp no. Passage MACS Media A253 1 CDM3 TH78 2 MAM6−CDM3 TH82* 3 ESA+ Biopsy A269 (TH95, primary suprabasal cells) Exp no.Passage MACS Media A269 1 CDM3 TH90 2 MAM6− CDM3 TH95* 3 ESA+ABREVIATIONS USED IN TABLE 3:Exp no.: number of experiment.MACS: MiniMACS magnetic cell separation system.MAM6−: flow-through of an anti-sialomucin column.MAM6+: retention in an anti-sialomucin column.β4+: retention in a β4-integrin column.ESA+: retention in an anti-ESA column.§: DMEM/F-12 medium 1:1 supplemented with glutamine, 2 mM final.CDM3: CDM3 medium (Petersen and van Deurs 1987)CDM6: CDM6 medium (Pechoux et al. 1999)H14: H14 medium (Blaschke et al. 1994)CT: cholera toxin (10 ng/ml final).EGF: epidermal growth factor (100 ng/ml final).KGF: keratinocyte growth factor (10 μg/ml final).I: insulin (3 μg/ml final).H: hydrocortisone (1.4 × 10⁶ M final).FCS: fetal calf serum (E.C. approved, virus and mycoplasma tested).*After ESA purification cells were immediately embedded into 300 μlMatrigel ® and cultured for 2 weeks in CDM3 medium.

TABLE 4 Primary antibodies. antibody directed against antibodydescription (name and provider) keratin K18 F3006; Trichem Aps, Denmark,keratin K19 BA17, DAKO, Glostrup, Denmark, keratin K19 RCK108, DAKO,Glostrup, Denmark, sialomucin MAM6, clone 115D8, Biogenesis Ltd., Poole,UK, occludin OC-3F10, Zymed Laboratories, San Francisco, CA, polyclonalclaudin-1 Zymed Laboratories, San Francisco, CA, epithelial-specificantigen ESA; VU-1D9, NovoCastra, Newcastle upon Tyne, UK, E-cadherinHECD-1, kindly provided by Dr. Atsushi Ochiai, Tokio, Japan, Thy-1AS0-2, Dianova, Hamburg, GmbH, α-smooth muscle actin 1A4, Sigma-Aldrich,Vallensbæk, Denmark, vimentin V9, DAKO, Glostrup, Denmark, α1 chain oflaminin-1 EB7, kindly provided by I. Virtanen, University of Helsinki,keratin K14 LL002, NovoCastra, Newcastle upon Tyne, UK.

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1. An isolated cell derived from luminal epithelial cells of a mammarygland which is capable of proliferating and differentiating into cellsof mammary gland luminal epithelial and myoepithelial cell lineages saidisolated cell being capable of forming a cell culture comprising cellswhich are positive staining for the luminal epithelial marker ESA (ESA+)and negative staining for sialomucin (MUC−), so-called (ESA+/MUC−)cells.
 2. A cell according to claim 1, which is isolated from suprabasalluminal epithelial cells of the mammary gland.
 3. A cell according toclaim 2, which is a human cell.
 4. A cell according to claim 1, which isimmortalised.
 5. A cell population composed of cells according toclaim
 1. 6. An immortalised cell line derived from the cell of claim 4.7. An immortalised cell line according to claim 6, wherein theimmortalising step comprises transfecting the cells with a nucleic acidmolecule encoding an immortalising polypeptide.
 8. An immortalised cellline according to claim 7, wherein the immortalising step comprisestransfecting the cells with a nucleic acid molecule encoding apapillomavirus polypeptide selected from the group consisting of E6, E7and a nucleic acid molecule comprising E6 and E7.
 9. An immortalisedcell line according to claim 7, wherein the immortalising step comprisestransforming the cells with at least one retroviral vector including anexpression cassette comprising a nucleic acid molecule encoding apapillomavirus polypeptide selected from the group consisting of E6, E7and a nucleic acid molecule comprising E6 and E7, and selecting theimmortalised cells.
 10. An immortalised cell line according to claim 9,wherein the immortalising step is performed by transforming the cellswith retrovirus-containing supernatant from the PA317 LXSN HPV16E6E7cell line and selecting the immortalised cells.
 11. An immortalised cellline according to claim 6 that in culture is capable of formingbranching structures resembling terminal duct lobular units of themammary gland in morphology and/or by marker expression.
 12. Animmortalised cell line according to claim 6 which comprises cells thatare positive staining for the keratin K19.
 13. An immortalised cell lineaccording to claim 6 that is derived from a cell selected from the groupconsisting of a rodent cell, a porcine cell, a ruminant cell, a bovinecell, a caprine cell, a equine cell, a canine cell, a ovine cell, afeline cell and a primate cell.
 14. An immortalised cell line accordingto claim 13 that is selected from the group consisting of cells frommice, rats and rabbits.
 15. An immortalised cell line according toclaims 13 that is a human cell line.
 16. The immortalised cell lineaccording to claim 6 which is deposited in accordance with theprovisions of the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure atDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) andhas obtained the accession number DSM ACC
 2529. 17. A method forisolation of an at least bi-potent mammary gland tissue cell, comprisingthe steps of: (i) separating said tissue into two or more different celltypes (ii) culturing each of said different cell types under celldifferentiation conditions and (iii) selecting the cell type(s) thatis/are capable of differentiating into at least two morphologicallyand/or phenotypically different cell types.
 18. A method according toclaim 17 in which the at least bi-potent cell is a cell according to anisolated cell derived from luminal epithelial cells of a mammary glandwhich is capable of proliferating and differentiating into cells ofmammary gland luminal epithelial and myoepithelial cell lineages saidisolated cell being capable of forming a cell culture comprising cellswhich are positive staining for the luminal epithelial marker ESA (ESA+)and negative staining for sialomucin (MUC−), so-called (ESA+/MUC−)cells.
 19. A method for testing the toxic effect, if any, of a substanceon mammary gland epithelial cells, the method comprising: (i) culturingor maintaining the cells of claim 1 in a non-toxic medium; (ii) addingthe substance to be tested to the medium; and (iii) determining theresponse, if any, of the cells, including changes in cell growth rate,cell death rate, apoptosis, cell metabolism, inter- as well asintra-cellular communication, morphology, mRNA or protein expression andantigen expression.
 20. A method for testing the carcinogenic effect, ifany, of a substance on mammary gland epithelial cells, the methodcomprising: (i) culturing the cells of claim 1 in a growth medium whichmaintains the cells as non-transformed cells; (ii) adding the agent,compound or factor under test to the cell culture; and (iii) determiningthe neoplastic response, if any, of the so contacted cells by changes inmorphology, tumorigenicity in animals, mRNA expression and/or antigenexpression as well as other changes which is associated withcarcinogenicy.
 21. A method as claimed in claim 20, wherein thetumorigenicity test comprise the introduction of said treated cells intoan immune incompetent test animal.
 22. A method of testing the ability,if any, of a substance to modulate the differentiation of non-terminaldifferentiated mammary gland epithelial cells, the method comprising:(i) culturing or maintaining the cells of claim 1 in a medium which intiself does not modulate the differentiation; (ii) adding the substanceunder test to the cell culture; and (iii) determining thedifferentiation modulation responses, if any, of the so contacted cellsby changes in cell growth rate, cell death rate, apoptosis, cellmetabolism, inter- as well as intra-cellular communication, morphology,mRNA or protein expression or antigen expression as well as otherchanges which is associated with differentiation.
 23. A method forscreening a substance for its ability, if any, to interact with acellular protein, the method comprising: (i) transfecting a cell ofclaim 1 with a gene construct enabling transfected cells to express saidprotein; (ii) adding the substance to be tested to the cells; and (iii)determining the interaction, if any, with a cellular protein by changesin cell growth rate, cell death rate, apoptosis, cell metabolism, inter-as well as intra-cellular communication, morphology, mRNA or proteinexpression, antigen expression or other changes which either directly orindirectly is supposed to be associated with said protein.
 24. A methodaccording to claim 23 in which said cellular protein is selected fromthe group consisting of estrogen receptor-alpha, estrogen receptor-betaand progesterone receptor.
 25. A method of transplanting a vertebratehost with a cell according to claim 1, comprising the step ofintroducing the cell into the vertebrate host.
 26. A method of in vivoadministration of a protein or gene of interest to an individual in needthereof, comprising the step of transfecting the cell-population ofclaim 1 with a vector comprising DNA or RNA which expresses the proteinor gene of interest and introducing the transfected cell into saidindividual.
 27. Use a cell according to claim 1 to prevent and/or treatcellular debilitations, derangements and/or dysfunctions and/or otherdisease states in mammals, comprising administering to a mammal atherapeutically effective amount of said cells, or cells or tissuesderived therefrom.
 28. A method of tissue repair or transplantation inmammals, comprising administering to a mammal a therapeuticallyeffective amount of a cell according to claim 1, or cells or tissuesderived therefrom.
 29. A pharmaceutical composition comprising: atherapeutically effective amount of a cell according to claim 1, orcells or tissues derived therefrom; and a pharmaceutically acceptablecarrier.
 30. The pharmaceutical composition of claim 29 furthercomprising a proliferation factor or lineage commitment factor.
 31. Adiagnostic agent comprising the cell of claim 1, or any part thereof